Detection and molecular characterization of Cryptosporidium spp. in captive canaries (Serinus canaria) using different diagnostic methods

Abstract

This study used several diagnostic methods to examine the occurrence of and molecularly characterize Cryptosporidium spp. in captive canaries (Serinus canaria) in southern and southeastern Brazil. A total of 498 fecal samples were purified by centrifugal-flotation using Sheather's solution. Cryptosporidium spp. diagnosis was performed using three diagnostic methods: malachite green negative staining, nested PCR targeting the 18S rRNA gene, followed by sequencing the amplified fragments, and duplex real-time PCR targeting the 18S rRNA specific to detect Cryptosporidium galli and Cryptosporidium avian genotype III. The overall positivity for Cryptosporidium spp. (total samples positive in at least one protocol) from the microscopic analysis, nested PCR and duplex real-time PCR protocol results was 13.3% (66/498). The positivity rates were 2.0% (10/498) and 4.6% (23/498) for Cryptosporidium spp. by microscopy and nested PCR, respectively. Sequencing of 20 samples amplified by nested PCR identified C. galli (3.0%; 15/498), Cryptosporidium avian genotype I (0.8%; 4/498) and Cryptosporidium avium (0.2%; 1/498). Duplex real-time PCR revealed a positivity of 7.8% (39/498) for C. galli and 2.4% (12/498) for avian genotype III. Malachite green negative staining differed significantly from nested PCR in detecting Cryptosporidium spp. Duplex real-time PCR was more sensitive than nested PCR/sequencing for detecting gastric Cryptosporidium in canaries.Keywords: Cryptosporidiosis, birds, diagnosis, epidemiology. ResumoEste trabalho teve como objetivos determinar a ocorrência e realizar a caracterização molecular de Cryptosporidium spp. em 498 amostras fecais de canários (Serinus canaria) criados em cativeiro, utilizando três métodos de diagnóstico: análise microscópica pela coloração negativa com verde malaquita, nested PCR seguida de sequenciamento dos fragmentos amplificados e PCR duplex em tempo real específica para detecção de Cryptosporidium galli e Cryptosporidium genótipo III de aves. A positividade total para Cryptosporidium spp. (total de amostras positivas em pelo menos um método de diagnóstico) obtida pela análise microscópica, nested PCR e PCR duplex em tempo real foi de 13,3% (66/498). As taxas de positividade para Cryptosporidium spp. foram 2,0% (10/498) e 4,6% (23/498) por microscopia e nested PCR, respectivamente. O sequenciamento de 20 amostras amplificadas pela nested PCR identificou C. galli (3,0%; 15/498), Cryptosporidium genótipo I de aves (0,8%; 4/498) e Cryptosporidium avium (0,2%; 1/498). A PCR duplex em tempo real revelou positividade de 7,8% (39/498) para C. galli e 2,4% (12/498) para Cryptosporidium genótipo III de aves. A análise microscópica diferiu significativamente da nested PCR para detecção de Cryptosporidium spp. A PCR duplex em tempo real apresentou maior sensibilidade que a nested PCR/sequenciamento para detectar as espécies/genótipos gástricos de Cryptosporidium.

Cross-genera amplification and identification of Colpodella sp. with Cryptosporidium primers in fecal samples of zoo felids from northeast China

“…The PCR primers used in this study were previously known to have the ability to amplify a segment of the Cryptosporidium sp. 18S rRNA gene (Alkhaled, 2017;Ayinmode et al, 2018;Ayinmode et al, 2017;Camargo et al, 2018;Lucio et al, 2016). But our results revealed that Colpodella sp.…”

Cryptosporidium cf. avium in an inland-bearded dragon (Pogona vitticeps) – A case report and review of the literature

“…No pathology. Abe and Matsubara, 2015 C. avium Buderigar, Cockatiel P Melopsittacus undlates Nymphicus hollandicus China KM267556 none none unknown Zhang et al (2015) C. avium * Red-crowned parakeet, Chicken, Budgerigar P Cyanoramphus novaezealandiae, Gallus gallus , Melopsittacus undlates Czechia KU058875-78 KU058879-82 KU058883-86 none Holubová et al (2016) C. avium Canary P Serinus canaria Brazil MG832882 none none none Camargo et al (2018) C. avium * Cockatiel P , Chicken* Nymphicus hollandicus , Gallus gallus China JQ246415 JQ320301 JQ798893 none Cui et al (2018) C. avium * Mallard, Chicken, Pheasant W,A Anas platryrhynchos , Gallus gallus , Phasianus colchicus Czechia none none none none Holubová et al (2018) C. avium Red-crowned parakeet, Budgerigar P Cyanoramphus novaezealandiae, ...…”

First Detection of Cryptosporidium spp. in Migratory Whooper Swans (Cygnus cygnus) in China

“…Other studies have also reported much higher infection rates of Cryptosporidium in wild birds than this study, e.g., in wild captive psittacines in Brazil (10.64% 5/47), Java sparrows (Lonchura oryzivora) in northern China (13.42% 47/350), and North American red-winged blackbirds (Agelaius phoeniceus) (17.1% 12/70), and Canada geese (23.4% 49/209) in Ohio and Illinois [25][26][27][28]. Previous studies recorded Cryptosporidium infection rates in domestic birds of 2.3%-4.86% in Brazil [29][30][31] and 0.82%-8.1% in China [32][33][34][35][36]. These variations in the positive rate of Cryptosporidium in different studies may be attributable to population densities, feeding habits, and climate.…”